Foxp3+ regulatory T cells (Tregs) are a subset of CD4 T cells that suppress inflammatory responses and serve as key determinants of autoimmunity, cancer progression, allograft rejection, maternal-fetal tolerance, and chronic infection. A high density of Tregs in tumor lesions predicts a poor clinical outcome in many human tumors, and Tregs restrict anti-tumor responses in several pre-clinical cancer models. Current approaches to modulating Treg-mediated suppression in the clinical setting involve global depletion or functional ablation, and these treatments are inherently associated with a risk of autoimmunity. Because Treg suppressive function requires signaling through the antigen-specific T cell receptor (TCR), those Tregs that are actively restricting immunity in most disease scenarios probably represent a small subset of the total Tregs, those that are specific for certain expressed antigens. However, immunotherapeutic strategies that selectively target these antigen-specific Tregs have not been devised. Work in our lab in the mouse model of Mycobacterium tuberculosis (Mtb) infection has revealed that a population of thymically-derived Tregs specific for a Mtb-derived epitope expands in the lung draining lymph node and potently suppresses the developing adaptive immune response during early infection. This highly immunosuppressive Treg subpopulation does not persist, but is selectively eliminated by the host immune response in an IL-12p40-dependent manner while other T regs in the same lymph node continue to increase in numbers. Utilizing the experimental systems that we and our collaborators have developed to study antigen-specific Tregs during infection and cancer, we will perform a mechanistic dissection of this process. In Aim 1, we will identify the IL-12p40-dependent cytokine that drives Mtb-specific Treg contraction and determine whether this cytokine acts directly on the Tregs or mediates the effect via other cell types. We will also investigate the idea that the cytokine mediates Treg culling by inducing production of nitric oxide species. In Aim 2, we will determine the extent to which the identified IL-12p40-dependent pathways regulate prostate antigen-specific T regs during a mouse model of prostrate cancer, and determine whether experimental manipulation of these pathways can induce the specific elimination of prostate antigen-specific T regs. These studies will identify a molecular axis that s required for the culling of pathogen- specific Tregs and explore the impact of this pathway on tumor-associated Tregs in the context of cancer. This will open new avenues for future investigation, and raises the distinct possibility that this axis could be exploited therapeuticall to selectively and safely eliminate harmful Treg subpopulations in cancer and other chronic diseases in which antigen-specific Tregs restrict immunity.